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Simple and inexpensive ribosome profiling analysis of mRNA translation.

Publication ,  Journal Article
Reid, DW; Shenolikar, S; Nicchitta, CV
Published in: Methods
December 2015

The development and application of ribosome profiling has markedly advanced our understanding of ribosomes and mRNA translation. The experimental approach, which relies on deep sequencing of ribosome-protected mRNA fragments generated by treatment of polyribosomes with exogenous nucleases, provides a transcriptome-wide assessment of translation. The broad application of ribosome profiling has been slowed by the complexity and expense of the protocol. Here, we provide a simplified ribosome profiling method that uses micrococcal nuclease to generate ribosome footprints in crude cellular extracts, which are then purified simply by size selection via polyacrylamide gel electrophoresis. This simplification removes the laborious or expensive purification of ribosomes that has typically been used. This direct extraction method generates gene-level ribosome profiling data that are similar to a method that includes ribosome purification. This protocol should significantly ease the barrier to entry for research groups interested in employing ribosome profiling.

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Published In

Methods

DOI

EISSN

1095-9130

Publication Date

December 2015

Volume

91

Start / End Page

69 / 74

Location

United States

Related Subject Headings

  • Sequence Analysis, RNA
  • Ribosomes
  • RNA, Messenger
  • Protein Biosynthesis
  • Micrococcal Nuclease
  • Lymphocytes
  • Humans
  • High-Throughput Nucleotide Sequencing
  • 3101 Biochemistry and cell biology
  • 1103 Clinical Sciences
 

Citation

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Reid, D. W., Shenolikar, S., & Nicchitta, C. V. (2015). Simple and inexpensive ribosome profiling analysis of mRNA translation. Methods, 91, 69–74. https://doi.org/10.1016/j.ymeth.2015.07.003
Reid, David W., Shirish Shenolikar, and Christopher V. Nicchitta. “Simple and inexpensive ribosome profiling analysis of mRNA translation.Methods 91 (December 2015): 69–74. https://doi.org/10.1016/j.ymeth.2015.07.003.
Reid DW, Shenolikar S, Nicchitta CV. Simple and inexpensive ribosome profiling analysis of mRNA translation. Methods. 2015 Dec;91:69–74.
Reid, David W., et al. “Simple and inexpensive ribosome profiling analysis of mRNA translation.Methods, vol. 91, Dec. 2015, pp. 69–74. Pubmed, doi:10.1016/j.ymeth.2015.07.003.
Reid DW, Shenolikar S, Nicchitta CV. Simple and inexpensive ribosome profiling analysis of mRNA translation. Methods. 2015 Dec;91:69–74.
Journal cover image

Published In

Methods

DOI

EISSN

1095-9130

Publication Date

December 2015

Volume

91

Start / End Page

69 / 74

Location

United States

Related Subject Headings

  • Sequence Analysis, RNA
  • Ribosomes
  • RNA, Messenger
  • Protein Biosynthesis
  • Micrococcal Nuclease
  • Lymphocytes
  • Humans
  • High-Throughput Nucleotide Sequencing
  • 3101 Biochemistry and cell biology
  • 1103 Clinical Sciences